ABSTRACT
Islet transplantation is a promising treatment for type 1 diabetes, but despite the successes, existing challenges prevent widespread application. Ischemia, occurring during pancreas preservation and isolation, as well as after islet transplantation, decreases islet viability and function. We hypothesized that the liposomal delivery of adenosine triphosphate (ATP) could prevent the loss of cell viability during an ischemic insult. In this work we use a model ß cell line, INS-1 to probe the liposome/cell interactions and examined the ability of liposomes functionalized with the fibronectin-mimetic peptide PR_b to facilitate the delivery of ATP to ischemic ß cells. We demonstrate that PR_b increases the binding and internalization of liposomes to the ß cells. Unexpectedly, when comparing the ability of PR_b liposomes with and without ATP to protect INS-1 cells from ischemia we found that both formulations increased cell survival. By probing the functional activity of ischemic cells treated with PR_b functionalized liposomes with and without ATP we find that both lipids and ATP play a role in maintaining cell metabolic activity after an ischemic insult and preventing cell necrosis. This approach may be beneficial for preventing ischemia related damage to islet cells, especially in the organ preservation stage.
ABSTRACT
Islet transplantation is a promising treatment for type 1 diabetes. Recent studies have demonstrated that human islet allografts can restore insulin independence to patients with this disease. As islet isolation and immunotherapeutic techniques improve, the demand for this cell-based therapy will dictate the need for other sources of islets. Pig islets could provide an unlimited supply for xenotransplantation and have shown promise as an alternative to human islet allografts. However, stresses imposed during islet isolation and transplantation decrease islet viability, leading to loss of graft function. In this study, we investigated the ability of a fibronectin-mimetic peptide, PR_b, which specifically binds to the alpha(5)beta(1) integrin, to re-establish lost extracellular matrix (ECM) around isolated pig islets and increase internalization of liposomes. Confocal microscopy and Western blotting were used to show the presence of the integrin alpha(5)beta(1) on the pig islets on day 0 (day of isolation) as well as on different days of islet culture. Islets cultured in medium supplemented with free PR_b for 48 h were found to have increased levels of ECM fibronectin secretion compared to islets in normal culture conditions. Using confocal microscopy and flow cytometry, we found that PR_b peptide-amphiphile functionalized liposomes delivered to the pig islets internalized into the cells in a PR_b concentration dependent manner and nonfunctionalized liposomes showed minimal internalization. These studies proved that the fibronectin-mimetic peptide, PR_b, is an appropriate peptide bullet for applications involving alpha(5)beta(1) expressing pig islet cells. Fibronectin production stimulated through alpha(5)beta(1) PR_b binding may decrease apoptosis and therefore increase islet viability in culture. In addition, PR_b peptide-amphiphile functionalized liposomes may be used for targeted delivery of different agents to pig islet cells.
